Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
Bhat Kumble, V.
Bioactive sphingolipids are critical regulators of Merkel Cell Carcinoma cell proliferation and survival
PhD-Studium (Doctor of Philosophy); Humanmedizin; [ Dissertation ] Medical University of Graz; 2019. pp. 124
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- Autor*innen der Med Uni Graz:
- Betreuer*innen:
- Heinemann Akos
- Marsche Gunther
- Sattler Wolfgang
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- Abstract:
- Merkel cell carcinoma (MCC) is an aggressive metastatic neuroendocrine tumor of the skin. The incidence of MCC is 0.6 in 100,000 persons. The mortality rate is 33% which exceeds melanoma. Though immune checkpoint inhibitor antibodies are approved by the FDA, the drug is ineffective in 50% of MCC cases. MCC is mainly driven by clonally integrated Merkel cell polyomavirus (MCPyV) DNA, and UV mediated DNA damage. Nearly 80% of MCC cases are MCPyV+, and the viral large and small tumor antigens (LT and sT, respectively) were shown to be oncogenic. The mechanism of clonal integration and subsequent transformation into MCC tumors is not fully understood. To be able to develop mechanism-based targeted therapies it is essential to gain detailed insight into pathways that drive MCC. Bioactive Sphingolipids are an indispensable class of sphingolipids that regulate cancer cell formation, survival, and proliferation. Sphingosine 1-phosphate (S1P) and ceramide (Cer) are the most studied bioactive sphingolipids. S1P is a pro-proliferative molecule, and Cer is mostly pro-apoptotic. Maintaining the rheostat between S1P and Cer is vital for normal cell function. In contrast, cancer cells upregulate S1P synthesis and inhibit Cer formation to survive and proliferate. Enzymes that synthesize these bioactive lipids are dysregulated in cancer cells. Serine palmitoyltransferase (SPTLC1-3) is an enzyme that regulates the first step of S1P synthesis pathway, and sphingosine kinase 1 and 2 (SK1/2) are the enzymes that synthesize S1P by phosphorylating its precursor sphingosine. Both of these enzymes where shown to be upregulated in various cancer entities. We hypothesized that targeting the sphingomyelin-ceramide-S1P rheostat could represent a novel anticancer strategy in MCC. To test our hypothesis, we first characterized the expression of SPTLC1-3 and SK1/2 in 21 MCC tissue samples and 16 MCC cell lines. Further, transfection of MCPyV-LTA derived from MKL-1, MKL-2, and MS1 MCC cells into lung fibroblasts upregulated SK1 and SK2 transcription. Additionally, analysis of published microarray GEO datasets revealed upregulation of SK1 in MCC as compared to healthy skin tissue. Also, reanalysis of published RNA sequencing data showed that forced MCPyV-sT expression upregulates SPTLC1/2 and SK1/2 expression in lung fibroblasts. For in-vitro studies, we used the well-characterized MKL-1 and WaGa MCC cell lines and non-transformed human dermal fibroblasts (NHDF) as control cells. To test the importance of S1P synthesis for MCC survival and proliferation, we pharmacologically targeted SPT with myriocin and SK1/2 activity with SKI-II and ABC294640. Of these, SKI-II inhibits both kinases, while ABC294640 is SK2 specific. In addition, we also used the FDA approved drug FTY720/fingolimod during MCC proliferation experiments. Myriocin treatment decreased cellular ceramide, sphingomyelin (SM), and S1P concentrations while SKI-II treatment decreased S1P and SM but increased ceramide in MKL-1 and WaGa MCC cells. Myriocin, SKI-II, ABC294640, and FTY720 reduced cell viability and increased apoptosis and necrosis. Of importance, non-transformed NHDF cell viability was not affected by myriocin, SKI-II, and ABC294640 treatment while FTY720 induced apoptosis. S1P-enriched high-density lipoprotein (HDL) increased the viability of MKL1 and WaGa cells in comparison to native HDL. Furthermore, inhibition of SPTLC1-3 and SKI-II with myriocin and SKI-II decreased phosphorylation of AKT and increased caspase3 and PARP activity in MKL-1 and WaGa cells. In a pre-clinically more relevant setting we could demonstrate that myriocin and SKI-II treatment reduced MKL-1 and WaGa tumor xenograft growth and proliferation in the ex-ovo chorioallantoic membrane tumor model. In conclusion, we show that the S1P pathway is critical for MCC survival in in-vitro and ex-ovo condition and targeting the SM-Cer-S1P rheostat holds promise to interfere with MCC tumor growth and proliferation.